“This book offers readers an excellent opportunity to have a broad idea of the important characteristics of probiotic microorganisms, based on data from recent research. The text covers scientific aspects, including the characterization of the microorganisms employed as probiotics, the physiology of the human body, and the effect of their interaction on human health, to arrive at foods that can be used as vehicles for probiotics, to develop pharmaceutical forms of probiotics, and to find out ways, such as immobilization and microencapsulation, to improve probiotic survival. The book gives a good overview of the guidelines and regulations related to probiotics followed worldwide. It is a very well-written and scientifically based book, which will be certainly useful for undergraduate and graduate students and professionals in health- and food-related areas.”

Prof. Susana Marta Isay SaadUniversidade de São Paulo, Brazil

“If you are interested in probiotics, I highly recommend this book for its scientific basis, didactic teaching and accessible presentation.”

Prof. Carmen Sílvia Favaro-TrindadeUniversidade de São Paulo, Brazil

The interest in probiotic microorganisms and the research on them date back to the early 20th century. The literature on this subject is vast and diverse and generally focuses on the benefits of probiotic microorganisms for the host. The stimulation of the immune response, improvement of the immunological barrier, higher resistance to microbial pathogens, and a positive metabolic contribution are some of the benefits suggested by several studies on probiotics.

This book is a unique compilation of technological aspects related to probiotics, in terms of not only delivery modes but also protection technologies, and includes their therapeutic and physiologic implications and benefits. It provides a contemporary update and holistic review of the topic and is designed to augment related books in the market. The novelty of this book is that it focuses on the technological aspects of probiotic products without neglecting their therapeutic role, brings together the information needed for the successful development of probiotic products, and examines the international picture regarding regulatory issues.

J. Paulo Sousa e Silva is assistant professor at the Faculty of Pharmacy, University of Porto, Portugal. He has headed the Pharmaceutical Development Division of Bial, Portugal, and participated as investigator in the project PROBIOCAPS—tailored microencapsulation technology for probiotic bacteria: production, stability and functionality enhancements. Currently, his research interests are focused on the development of drug delivery systems, microencapsulation, and nanoencapsulation.

Ana C. Freitas is a food engineer. She received her PhD in biotechnology from the Catholic University of Portugal in 1999. Since then, she is assistant professor at the Instituto Piaget, Portugal. From 2011, she has also been a postdoctoral researcher at CESAM, University of Aveiro, Portugal. She was the principal investigator of the project PROBIOCAPS. Currently, her research is focused on the development of novel foods incorporating bioactive compounds derived from natural sources.

ISBN 978-981-4411-62-2V296

Sousa e S

ilva | FreitasProbiotic Bacteria Fundamentals, Therapy, and Technological Aspects

edited by

J. Paulo Sousa e SilvaAna C. Freitas

Probiotic Bacteria

Probiotic Bacteria

for the WorldWind PowerThe Rise of Modern Wind Energy

Preben MaegaardAnna KrenzWolfgang Palz

editorsFundamentals, Therapy, and Technological Aspects

edited by

J. Paulo Sousa e SilvaAna C. Freitas

Probiotic Bacteria

Published by

Pan Stanford Publishing Pte. Ltd.Penthouse Level, Suntec Tower 3 8 Temasek Boulevard Singapore 038988

Email: editorial@panstanford.com Web: www.panstanford.com

British Library Cataloguing-in-Publication DataA catalogue record for this book is available from the British Library.

Probiotic Bacteria: Fundamentals, Therapy, and Technological AspectsCopyright © 2014 by Pan Stanford Publishing Pte. Ltd.All rights reserved. This book, or parts thereof, may not be reproduced in any form or by any means, electronic or mechanical, including photocopying, recording or any information storage and retrieval system now known or to be invented, without written permission from the publisher.

For photocopying of material in this volume, please pay a copying fee through the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA. In this case permission to photocopy is not required from the publisher.

ISBN 978-981-4411-62-2 (Hardcover)ISBN 978-981-4411-63-9 (eBook)

Printed in the USA

Contents

Foreword ixPreface xi

1. Probiotic Bacteria: From Science to Consumers’ Benefit 1

Manuela M. Pintado, Ana M. Gomes, and Ana C. Freitas

2. Gastrointestinal Tract: Microflora and Transit Aspects 7

Ana M. Gomes, Manuela M. Pintado, Ana C. Freitas, and J. Paulo Sousa e Silva

2.1 Gastrointestinal Physiology 7 2.1.1 Introduction 7 2.1.2 GIT pH 9 2.1.3 Total Residence Time in the GIT 10 2.1.4 Mouth and Oesophagus 10 2.1.5 Stomach 11 2.1.6 Small Intestine 11 2.1.7 Large Intestine 12 2.2 GutMicroflora:Human‘VirtualOrgan’ 12 2.2.1 Ecological Niches 12 2.2.2 PhysiologicalRoleofGutMicroflora 15 2.2.2.1 Antagonistic mechanisms:

barrier function 16 2.2.2.2 Metabolic contribution 17 2.2.2.3 Immunomodulation 21 2.2.3 Balance between Detrimental/

BeneficialGutMicrobiotaand Intestinal Disorders 22

2.3 Selection of Target Organisms 26 2.3.1 ProbioticsandTheirEffectsUponGut

Microbiota 26 2.3.2 GIT In vitro Models for Selection 28 2.4 Conclusions 33

vi Contents

3. Probiotics and Their Therapeutic Role 47

Manuela M. Pintado, Ana M. Gomes, and Ana C. Freitas 3.1 Health Potential of Probiotics: Mechanisms

of Action 47 3.2 Alleviation of Lactose Intolerance 52 3.3 Inhibition of Helicobacter pylori and

Intestinal Pathogens 55 3.3.1 Inhibition of Helicobacter pylori 55 3.3.2 Inhibition of Other Enteric Pathogenic

Bacteria and Prevention and Reduction of Diarrhea Symptoms 60

3.3.3 InhibitionofEntericVirus:Rotavirus 63 3.4 PreventionofInflammatoryBowelDisease 66 3.5 HypocholesterolemicEffect 70 3.6 Treatment and Prevention of Allergy 72 3.7 Reduction of the Risk Associated with

Mutagenicity and Carcinogenicity 75 3.8 EffectonUrogenitalInfections 79 3.9 BenefitsfortheHealthyFunctionoftheLiver

and Pancreas 81 3.10 Oral Health–Promoting Properties 83 3.11 Other Therapeutic Applications 84 3.12 Conclusions 85

4. Food as Vehicles of Probiotics 95

Ana C. Freitas, Dina Rodrigues, Sérgio Sousa, Ana M. Gomes, and Manuela M. Pintado

4.1 Introduction 95 4.2 Dairy Products 96 4.2.1 Fermented Milks 96 4.2.1.1 Definition,classification,

market, and physiological role 97 4.2.1.2 Technological challenges for

probiotic fermented milks 101 4.2.1.3 Survival characteristics 114 4.2.2 Cheeses 115 4.2.2.1 Strains, cell probiotic

concentration, and viability 116 4.2.2.2 Cheese: Technological

aspects 128

viiContents

4.3 Nondairy Products 134 4.3.1 Beverages 134 4.3.2 Other Products 144 4.4 Conclusions 151

5. Immobilization and Microencapsulation of Probiotics 171

Paulo J. C. Costa, Teresa Rocha-Santos, Ana M. Gomes, Manuela M. Pintado, Sérgio Sousa, Maria H. Amaral, J. Paulo Sousa e Silva, and Ana C. Freitas

5.1 Introduction 171 5.2 Microencapsulation 175 5.2.1 Encapsulating Materials 176 5.3 Methods of Microencapsulation 178 5.3.1 Microparticle Formation 178 5.3.1.1 Liquid matrix dispersion 178 5.3.1.2 Solid matrix techniques 194 5.3.2 Immobilization/Entrapment

Techniques 197 5.3.2.1 Solidification 197 5.3.2.2 Coacervation 198 5.3.2.3 Gelation 201 5.3.2.4 Solvent extraction/

evaporation 203 5.3.2.5 Polymerization 205 5.3.3 Other Methods 206 5.4 Microparticle Characterization 208 5.5 Conclusions and Future Trends 209

6. Development of Probiotic Dosage Forms 227

Maria H. Amaral, J. Paulo Sousa e Silva, Paulo J. C. Costa, and Ana M. Gomes

6.1 Introduction 227 6.2 Manufacturing of Dosage Forms Containing

Probiotics 230 6.2.1 Powders 233 6.2.2 Capsules 234 6.2.3 Tablets 236 6.2.4 VaginalSuppositories 240 6.2.5 Other Pharmaceutical Dosage Forms 243 6.2.5.1 Chewing gums and lozenges 243

viii Contents

6.2.5.2 Gels 245 6.2.5.3 Eye drops 246 6.2.5.4 Pellets 246 6.3 Dosage Forms Characterization 247 6.4 Packaging and Storage of Dosage Forms

Containing Probiotics 253 6.5 Conclusions and Future Trends 254

7. Guidelines and Regulations 263

J. Paulo Sousa e Silva and Ana M. Gomes 7.1 Introduction 263 7.1.1 Food Standards 264 7.1.2 Drug Standards 265 7.2 Guidelines for Probiotic Selection 265 7.2.1 Safety Criteria 266 7.2.2 Functionality, Technological, and

Labeling Criteria 268 7.3 Probiotics Legal Status 272 7.3.1 Asia–Pacific 272 7.3.1.1 Australia and New Zealand 272 7.3.1.2 China 275 7.3.1.3 Japan 277 7.3.2 Europe 281 7.3.2.1 Central legislation 283 7.3.2.2 Web pages 283 7.3.3 United States of America 283 7.3.3.1 Legislation 286 7.3.3.2 Web pages 287 7.3.4 Latin America (Brazil) 287 7.3.4.1 Legislation 289 7.4 Conclusions 290

Index 295

ixContents

It gives me immense pleasure to write a few words about the upcoming book Probiotic Bacteria: Fundamentals, Therapy, and Technological Aspects edited by Drs. J. Paulo Sousa e Silva and Ana Cristina Freitas. Probiotic organisms have been a subject of keen research for more than a century. Studies on probiotic organisms have come a long way from the era of Metchnikoff in the early1900s, and our knowledge is being enhanced with each passing decade. Traditionally, probiotic organisms have been incorporated in fermented products like yogurt, however, as of recent times there are numerous probiotic products in the global market of varied types. The literature on probiotic organisms is vast and diverse and there is enough clinical evidence to support the health-enhancing potential of probiotic organisms. This book is a unique compilation of technological aspects related toprobioticproducts, theirbenefits, and their therapeuticand physiological implications. The information is conveniently grouped under seven chapters. Chapter 1 details the fundamentals of probiotic bacteria. Chapter 2 covers the gastrointestinal physiology and its relevance to probiotic products. Chapter 3 deals with the various therapeutic roles of probiotics beyond gut-health. Chapter 4 gives an overview on the advances in probiotic food, with judicious insights into the technological and functional aspects. Chapter 5 provides a detailed analysis of several materials and techniques for immobilization and microencapsulation of probiotic bacteria. Chapter 6 is devoted to the development of probiotic dosage forms, and Chapter 7 focuses on the guidelines and regulation pertaining to the use of probiotic organisms. In general, an attempt has been made to provide a comprehensive review on the fundamentals of probiotic organisms, along with their therapeutic and industrial aspects. The book is unique in presenting a dedicated section on the development of several dosage forms containing probiotic bacteria. The book provides a contemporary update and a holistic review of the topic, and is designed to augment related books in the market. The editorial team comprises individuals

Foreword

x

withnoteworthyandremarkableexperienceinthefieldofprobioticorganisms. It is anticipated that this book should be an indispensable resourceforacademicians,extensionstaff,andstudentsworkinginthe field of probiotic organisms and probiotic products. Also, thebook should appeal to technologists and food scientists in the related industry.

Nagendra P. Shah

Professor of Food Science

School of Biological Sciences

The University of Hong Kong, Hong Kong

Foreword

xiContents

Probiotic organisms, according to the Food and Agriculture Organization (FAO) of the United Nations and the World Health Organization (WHO), are live microorganisms that when administeredinadequateamountscouldconferahealthbenefitonthe host. Probiotics can play a major role in human health if they can be incorporated in food products or used as dosage forms. For this, a detailed knowledge of the microorganisms is required, which forms the basis of the selection and use of probiotics. Probiotics may be useful in several functions, namely protection against pathogenic bacteria directly via displacement of these bacteria by competitive binding or growth inhibition, by antimicrobial compounds or pH reduction, or indirectly by neutralization or elimination of toxins from the intestine, improving gut barrier integrity by ameliorating epithelial and tissue integrity through low-dose NO synthesis, simulation of mucus production, or/and enhancing gut epithelial cell proliferation. In addition, probiotics have also been shown to have immunomodulation capacity, to inhibit endogenous carcinogen production, and to provide nutrients for enterocytes by short-chain fatty acid production. Such activities by a specific strain (or group) allow them to promote severalhealth benefits, which enables assuring of different therapeuticapplications, including alleviation of lactose intolerance, inhibition of Helicobacter pylori or other enteric pathogenic bacteria and enteric virus (particularly Rotavirus) with reduction of associated diarrhoea symptoms,preventionofinflammatoryboweldisease,reductionofcholesterol level, treatment and prevention of allergy, reduction of the risk associated with mutagenicity and carcinogenicity, reduction and control of urogenital infections, improvement in liver and pancreas dysfunctions, promotion of oral health. This book, organized in seven chapters, will help to understand what a probiotic is, how to isolate and assess the efficiency andsafety of each strain, and to elucidate about health benefits andmain mechanisms of action presenting the major current in in vitro, animal, and human studies supporting these properties.

Preface

xii Preface

Chapter 1 introduces the theme and summarizes the steps of launching probiotic products in the market. Chapter 2 presents an updated overview of the human intestinal microbial ecosystem from both endogenous and exogenous perspectives. At an endogenous level, the chapter covers the available knowledge on the dominant microbiota composition and stability, discusses the functional roles bacteria play in human health and well-being, and analyzes the consequences of homeostasis rupture among microbial balance as far as intestinal disorders are concerned. At the exogenous level, the chapter illustrates that target organisms are capable of modulating gutmicrobiotaandofpromotingdifferentphysiologicalroles.Thechapter also gives perspectives on the use of probiotics in dietary management and disease risk reduction. The main goal of Chapter 3 is to describe the relevant health potential of probiotics and current advances. The beneficialproperties assigned to probiotics and the corresponding specificmechanisms of action that will support each of the subsequent therapeutic applications will be explored in this chapter. Chapter 4 aims to provide a comprehensive overview on the advances in probiotic food, covering the technological issues, functionality aspects, and limitations of some foods as carriers of probiotics. This chapter is divided in two parts: the first partcovers dairy products, fermented milks, and cheeses that constitute the major group of products that can carry and deliver probiotic bacteria; the second part covers non-dairy products where alternative functional foods with probiotics such as juices and other food carriers are presented and discussed. Toconferhealthbenefitstothehumanhost,probioticsmustbekeptaliveuntiltheyreachtheirsiteofaction.InChapter5differentapproaches including immobilization and/or encapsulation of probiotics inside a protective material in order to increase the resistance of these sensitive microorganisms against adverse conditions have been revisited. Theprobioticstrainsintendedtobenefithealthortreat illnessmay be incorporated into suitable dosage forms in which they can maintain their effectiveness. Therefore, Chapter 6 is concernedwith dosage forms, such as oral powders, capsules, oral and vaginal tablets, vaginal suppositories, chewing gums, gels, eye drops, and pellets, that are used to administer probiotics. The definition of

xiiiPreface

these dosage forms, their respective processes of manufacturing, and characterization tests are also addressed in this chapter. In Chapter 7, which is the last chapter of the book, the global legal framework for probiotics is addressed. Generically, probiotics may be considered as a food, including food additives and dietary supplements, or as a drug. The chapter underlines the safety considerations and presents a comprehensive report on all necessary requirements related to them. In summary, the book intends to provide a comprehensive overview of the fundamental concepts, mechanisms, therapeutic actions, technological aspects, and ongoing research related with probiotic bacteria. The book will be helpful for students and scientists from the food science and technology, pharmacy, and nutrition sciences fields; scientists working in the field of gastrointestinaldisorders and other chronic diseases; companies who are designing and marketing new functional foods or nutraceuticals; as well as other public health professionals and clinicians. Furthermore, it provides important information for all readers interested in the relationship between food and health. This scientific work was a team effort written by a group ofscientistsfromthefoodandpharmaceuticalresearchfieldsdirectlyinvolved in the development of project PROBIOCAPS (PTDC/AGR-ALI/71051/2006; FCOMP-01-0124-FEDER-008792): Ana Gomes, Dina Rodrigues, Helena Amaral, Manuela Pintado, Paulo Costa, Sérgio Sousa, Teresa Rocha-Santos, and the editors of this book, and through individual research grants (SFRH/BD/77647/2011; SFRH/BPD/73781/2010; SFRH/BPD/65410/2009) by FCT. We would like to thank all these scientists for their contribution, and all otherswho,indifferentareasandskills,helpedthisprojectbecomea real success. We would also like to thank the reviewers for their professional advice and reviewing the chapters of this book.

J. Paulo Sousa e SilvaAna C. Freitas

Winter 2013